Reagent and method for bilirubin determination

ABSTRACT

A reagent and method for the determination of total serum bilirubin by coupling of bilirubin with a stable diazonium salt, Fast Red PDC or diazotate-N&#39;&#39;-butyl-4-methoxy-metanilamide, at an acid pH is disclosed.

(111 3,825,411 [45] July 23, 1974 REAGENT AND METHOD FOR BILIRUBIN DETERMINATION [75] Inventor: Leo G. Morin, Miami, Fla.

[73] Assignee: Medico Electronic, Inc.,

Indianapolis, Ind.

[22] Filed: Aug. 30, 1972 [21] Appl. No.: 284,883

[52] US. Cl. 23/230 B, 252/408 [51] Int. Cl. G0ln 33/16 [58] Field of Search 23/230 B; 252/408 [56] References Cited UNITED STATES PATENTS 2,854,317 9/1958 Free et al. 23/230 3 5/1970 Green 23/230 B 6/1971 Mast 23/230 B Primary Examiner-R. E. Serwin Attorney, Agent, qr Firm-Christen & Sabol; Virgil H. Marsh [57 ABSTRACT A reagent and method for the determination of total serum bilirubin by coupling of bilirubin with a stable diazonium salt, Fast Red PDC or diazotate-N'-butyl-4- methoxy-metanilamide, at an acid pH is disclosed.

14 Claims, No Drawings BACKGROUND OF THIS INVENTION 1. Field of this Invention This invention relates to a novel reagent and method for the colorimetric determination of total serum bilirubin; and more particularly, to a novel method for the determination of bilirubin by coupling with a specific diazonium salt, diazotate-N'-butyl-4- methoxymetanilamide at an acid pH.

2. Prior Art second approach suffers from the instability of reagents and requiresthat the diazotized sulfanilic acid be prepared fresh daily; The third approach has heretofore suffered from insensitivity and all methods using stable diazonium salts have been qualitative and semi quantitative at best, rather than quantitative. There is a need for a method to determine bilirubin that is specific and not subject to interference, and which utilizes a reagent that is reasonably stable and does not require daily preparation.

BROAD DESCRIPTION OF THIS INVENTION It is the primary objective of this invention to provide a colorimetric method and reagent for the determination of bilirubin. 1

Another object is to provide a direct methodwithout the need to remove proteins and possible interfering substances.

Still another object is to provide a method that utilizes a stable reagent that need not be prepared fresh dail Y t another object is to provide a method that is highly sensitive and specific, yielding quantitative determinations.

It has been found that a low pH (of 0.5 to 2.5, preferably about 1.8) in a solution of high molarity diazonium salts such as Fast Red RC, Fast Red GG, Fast Red B, Fast Red TR, Fast Blue B, Fast Bordeaux BD, etc., couple with bilirubin to give a color change that can be used to assy bilirubin. Of the innumerable diazonium salts tested, Fast Red PDC (diazoate-N'-butyl-4- methoxymetanilamide) proved to have significantly greater sensitivity, specificity, and extinction than the others. In addition, it was found that assays with Fast Red PDC were quantitative and not merely qualitative or semi-quantitative. So the preferred diazonium salt is Fast Red PDC. It has further been found that maleic acid is superior as a buffer in such bilirubin determinations to other acids that buffer in the desired pH range of about 0.5 to about 2.5. Such other acid buffers include oxalic acid, trihydroxybenzoic acid, dihydroxy- 2 tartaric acid, citric acid, fumaric acid, succinic acid, hydrochloric acid, sulfuric acid, butyric acid, dichloroacetic acid, hihydroxymalic acid, malonic acid, lactic acid, boric acid, etc.

The first embodiment of this invention involves a teagent which is a diazonium salt and maleic acid buffer,

the solution having a pH between 0.5 and 2.5.

The reagent, in a more specific sense, contains 0.01 to 10 gram percent of a diazonium salt 01' Fast Red PDC, and 5 to 50 gram percent of a suitable acid buffer or maleic acid buffer (preferably 22 percent maleic acid buffer). The reagent preferably has a pH of 1.8.

This invention is practised by mixing a small sample of biological fluid with a reagent, in a ratio of 1/ to l/ 25. The absorbance is read, using a colorimeter, at an appropriate wavelength or with a suitable color filter. With Fast Red PDC, the absorbance, is read between 490 and 650 nm, preferably at 590 nm. The absorbance is then converted to bilirubin concentration, such conversion can be done by means of preparing a conversion chart from known examples as illustrated in Exam- 7 ple l.

The reagent of this invention does not have to be prepared daily and can be in effect freeze-dried for storage of up to one year before use. The method of this invention is highly sensitive and specific, and yields quantitative determination.

Any suitable colorimeter or spectrophotometer can be used to measure the absorbance. Examples of useful colorimeters are: Coleman, Model 44; Perkin-Elmer, Model 124; the colorimeter disclosed in US. Ser. No. 224,457, applicantsi Raymond W. Kiess and Peter H. Stewart, filed: Feb. I 8, 1972, assignee: Kiess Instruments, Inc., 8768 S. W. 131st Street, Miami, Florida, 33156; and the direct reading colorimeter disclosed in US. Pat. 'No. 3,561,878, inventor: R. W. Kiess.

The term acid buffer, as used herein, means that a salt thereof is included. An example is maleic acid buffer, which includes maleic acid and a salt or ester thereof, e.g., sodium maleate, potassium maleate, barium maleate and lithium maleate.

' Examples of useful diazonium salts are Fast Red PDC (preferred), Fast Red RC, Fast Red 66, Fast Orange GR, Fast Red B, Fast Orange R, Fast Red TR, Fast Red base AL, Fast Blue B, Fast Red B base, Fast Bordeaux BD, Fast Red GG base, Fast Black B base, Fast Red GL base, Fast Blue base, Fast Red 3 GL base, Fast Blue Red 0 base, Fast Red RL base, Fast Scarlet G base, and fast Scarlet R base.

The biological fluids tested can'be those of man or animal.

DETAILED DESCRIPTION OF THIS INVENTION leic acid are added and dissolved, and the reagent is brought with water to 1 liter. To a series of tubes is added 2.5 ml of reagent. A set of standards. are prepared and made to contain 0.1, 0.5, 0.8, 1.0. 204.0,

12.0 and 15.0 mg percent of a specified level of bilirubin. The tubes are incubated at 37 C. for 5 minutes. A stable purple color develops and the absorbance is read at 590 nm using a colorimeter (Coleman Model 44).

EXAMPLE 2 The reagent is prepared and tubed as in Example 1. A set of standards are prepared as in Example 1, except that they are prepared in pooled human serum. The'test procedure of Example 1 is followed. Again, a stable purple color develops within minutes. The absorbance is'linear in proportion to the bilirubin concentration.

EXAMPLE 3 EXAMPLE 4 The reagent is prepared into two components. The first component is prepared by dissolving grams of Fast Red PDC in 1 liter of deionized water, dispensing 0.5 ml to a set of ampules, freeze-drying the dye in the ampules, and sealing the ampules under nitrogen. The second component is prepared by dissolving 220 grams of maleic acid in a'liter of deionized water. The two components are stored at room temperature for one year. At the end of a year, 2.5 ml of the second component is added to the tubes of the freeze-dried first component. The reagent is then tested as in Example 2. The results are the same as in Example 2.

What is claimed is:

l. A reagent for determining total bilirubin in biological fluids comprised of an aqueous solution of Fast Red PDC, which is diazoate-N,-butyl-4- methoxymetanilamide, and an acid buffer, said solution having a pH between 0.5 and 2.5.

2. A reagent according to claim 1 wherein the acid buffer is maleic acid buffer.

' 3. A reagent according to claim 1 wherein the Fast Red PDC is present at a level of 0.01 to 10 gramvpercent.

4. A reagent according to claim 1 whereinthe acid buffer is present at a level of 5 to 50 percent .by weight or volume.

5. A reagent according to claim 1 wherein the Fast Red PDC is present at a level of 0.01 to 10 gram percent, and the acid buffer is present at a level of 5 to 50 percent by weight.

6. A reagent according to claim 5 wherein the acid buffer is maleic acid buffer.

7. A reagent according to claim 6 wherein themaleic acid buffer includes maleic acid and a member selected from the group consisting of sodium maleate, potassium maleate, barium maleate, and lithium maleate.

8. A method for determining the total bilirubin in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 19, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of bilirubin by means of measuring the absorbance.

9. A method according to claim 8 wherein the acid buffer is maleic acid buffer.

10. A method according to claim 8 wherein the Fast Red PDC is present at a level of 0.01 to 10 gram percent.

11. A method according to claim 8 wherein the acid is present at a level of 5 to 50 percent by weight or volume.

12. A method according to claim 8 wherein the Fast Red PDC is present at a level of 0.01 to 10 gram percent, and the acid buffer is present at a level of 5 to 50 percent by weight or volume.

13. A method according to claim 12 wherein the acid buffer is maleic acid buffer.

14. A method according to claim 13 wherein the maleic acid buffer includes maleic acid and a member selected from the group consisting of sodium maleate, potassium maleate, barium maleate, and lithium maleate. 

2. A reagent according to claim 1 wherein the acid buffer is maleic acid buffer.
 3. A reagent according to claim 1 wherein the Fast Red PDC is present at a level of 0.01 to 10 gram percent.
 4. A reagent according to claim 1 wherein the acid buffer is present at a level of 5 to 50 percent by weight or volume.
 5. A reagent according to claim 1 wherein the Fast Red PDC is present at a level of 0.01 to 10 gram percent, and the acid buffer is present at a level of 5 to 50 percent by weight.
 6. A reagent according to claim 5 wherein the acid buffer is maleic acid buffer.
 7. A reagent according to claim 6 wherein the maleic acid buffer includes maleic acid and a member selected from the group consisting of sodium maleate, potassium maleate, barium maleate, and lithium maleate.
 8. A method for determining the total bilirubin in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 19, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of bilirubin by means of measuring the absorbance.
 9. A method according to claim 8 wherein the acid buffer is maleic acid buffer.
 10. A method according to claim 8 wherein the Fast Red PDC is present at a level of 0.01 to 10 gram percent.
 11. A method according to claim 8 wherein the acid is present at a level of 5 to 50 percent by weight or volume.
 12. A method according to claim 8 wherein the Fast Red PDC is present at a level of 0.01 to 10 gram percent, and the acid buffer is present at a level of 5 to 50 percent by weight or volume.
 13. A method according to claim 12 wherein the acid buffer is maleic acid buffer.
 14. A method according to claim 13 wherein the maleic acid buffer includes maleic acid and a member selected from the group consisting of sodium maleate, potassium maleate, barium maleate, and lithium maleate. 